1. Field of the Invention
This invention relates to an improved method for chelating multivalent cations. More particularly, the invention relates to a method of synthesizing photolabile chelators for chelating multivalent cations to be used for rapid delivery of such cations within biologic systems.
2. Description of the Prior Art
Calcium plays an important role as a "secondary messenger" in a variety of physiological processes in the regulation of cellular Ca.sup.2+ and it is central to the control of excitation-contraction coupling in muscle and of excitation-secretion coupling in many systems. In addition, magnesium is a necessary cofactor in ATP-dependent enzymatic processes including ion pumps, the myosin ATPase in muscle and a variety of kinases. It is also necessary in most processes where organo phosphates are enzymatic substrates. Therefore, the ability to regulate rapidly the concentration of the divalent cations of these metals for the quantitative study of the kinetics of such processes with minimal perturbation to the system is desirable.
One of the prime applications for optical indicators in the field of biology is a study of how calcium ions act as intracellular signals. As stated above, fluctuations in cytosolic free calcium concentrations [Ca.sup.2+ ]are hypothesized to be crucial in the triggering and control of a wide variety of cellular responses. To test such hypotheses, one would ideally verify first that, during a physiological response, the [Ca.sup.2+ ]does change with an amplitude and time course consistent with a triggering role. Assuming that a rise in Ca.sup.2+ concentration is detected, another important test is to raise the cytosolic [Ca.sup.2+ ]by artificial experimental means and see whether or not the physiological response is elicited.
The responses to calcium and magnesium in biological systems are often very rapid. Therefore, in order to adequately study such systems and the affect of such elements on them, a quick delivery system is required. Traditional methods for raising the Ca.sup.2+ concentration have included ionophore administration or direct microinjection or ionophoresis of Ca.sup.2+. Each of these methods have their problems, however, the principal one being the inability to raise the Ca.sup.2+ concentration as rapidly as is desired.
Recently, a new approach has developed that uses optical probes in reverse. In such a process, photochemically sensitive chelators directed to particular cations are synthesized. These chelators can then change irreversibly from high to low cation affinity upon illumination. Thus, light releases the "caged" cation and generates a jump in cation concentration. In general, such a method should be more controllable in amplitude, time course, and spatial extent than is currently achievable by other classical techniques. The chief advantage of this light flash technique is the speed of the photochemistry and the fact that it can be applied to organized systems, such as muscle fibers and membranes under electrophysiological investigation, that cannot be flowed.
Several caged compounds have been developed in the field of light flash physiology. A general overview of that field is provided in A.M. Gurney & H.A. Lester, Light-flash Physiology With Synthetic Photosensitive Compounds, 67(2) PHYSIOLOGICAL REVIEWS (April, 1987), incorporated herein by reference.
One general approach to the rapid release of physiologically significant organic molecules and cations is the "caging" of their active functionality using the photoremovable ortho-nitrobenzyl group. This approach has been utilized to cage ATP where a 2-nitrobenzyl ester of ATP releases ATP upon illumination.
Several variations on caged calcium chelators have also been developed. One example, referred to as nitr-2, is discussed 40 OPTICAL METHODS IN CELL PHYSIOLOGY, CHAPTER 19, "New Tetracarboxylate Chelators for Fluorescence Measurement and Photochemical Manipulation of Cytosolic Free Calcium," R.Y. Tsien (1986). Photolysis leads to the breakoff of MeOH, thereby decreasing the calcium binding affinity of the rest of the molecule and uncaging the Ca.sup.2+.
Nitr-2 effects a change in its effective dissociation constant from near 170 .mu.M to 7 .mu.M upon irradiation. In other words, nitr-2 binds calcium approximately 41 times as strongly prior to irradiation. While this compound marks a significant step forward, the magnitude of the change in calcium binding affinity is not as great as would be preferred. Further, the quantum yield upon irradiation is in the neighborhood of only 0.03 to 0.1. In addition, because the drop in calcium affinity is only on the order of 41 times, not all of the calcium originally caged to the now-cleaved molecules is released. Some of it remains chelated to the cleaved molecules. Thus, only a small amount of the caged calcium is released at any given time during irradiation. Even nitr-5, a revised version of nitr-2 referred to in the PHYSIOLOGICAL REVIEWS article cited above suffers from the same limitation on the change in calcium binding affinity. Still further, these prior art compounds are not suitable for caging other cations such, as Mg.sup.2+, that are of significant interest to biological researchers.
Accordingly, there exists a need for a photolabile chelator capable of caging multivalent cations and thereafter releasing large amounts of such multivalent cations upon irradiation.